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AAT Bioquest/Buccutite&trade；过氧化物酶（HRP）抗体结合试剂盒*优化用于标记100μg蛋白质*/5503/1试剂盒
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | R |
| Category | ProteinBiochemistry Generalproteins |
| Related |
Uponreceipt,storethekitat4oC.Whenstoredproperly,thekitshouldbestableforsixmonths.AlternativelyComponentsAandBcanbestoredat-20°C.DonotfreezeReactionBuffer(ComponentC)andSpinColumn(ComponentD).Warmallthecomponentsandcentrifugethevialsbrieflybeforeopening,andimmediatelypreparetherequiredsolutionsbeforestartingyourconjugation.ThefollowingSOPisanexampleforlabelinggoatanti-mouseIgGantibody.
1.Prepareantibodysolution:
Forlabeling100µgantibody(assumingthetargetantibodyconcentrationis1mg/mL),mix5µL(5%ofthetotalreactionvolume)ofReactionBuffer(ComponentC)with100µLofthetargetantibodysolution.
Note1.Ifyouhaveadifference concentration,adjusttheantibodyvolumeaccordinglytomake~100µgantibodyavailableforyourlabelingreaction.
Note2:Theantibodyshouldbedissolvedin1Xphosphatebufferedsaline(PBS),pH7.2-7.4;Iftheantibodyisdissolvedinglycinebuffer,itmustbedialyzedagainst1XPBS,pH7.2-7.4,oruseAmiconUltra-0.5,Ultracel-10Membrane,10 kDa(cat#UFC501008fromMillipore)toremovefreeaminesorammoniumsalts(suchasammoniumsulfateandammoniumacetate)thatarewidelyusedforantibodyprecipitation.
Note3:ImpureantibodiesorantibodiesstABIlizedwithbovineserumalbumin(BSA)orgelatinwillnotbelabeledwell.
Note4:Theantibody–Buccutite™MTAreactionefficiencyissignificantlyreducediftheantibodyconcentrationislessthan1mg/mL.Foroptimallabelingefficiencythefinalantibodyconcentrationrangeof1-10mg/mLisrecommended.
2.RunAntibody-Buccutite™MTAreaction:
2.1 AddtheantibodysolutiondirectlyintothevialofBuccutite™MTA(ComponentB),andmixthemwellbyrepeatedlypipettingforafewtimesorvortexthevialforafewseconds.
2.2 Keeptheantibody-Buccutite™MTAreactionmixtureatroomtemperaturefor30-60minutes.
Note:Theantibody-Buccutite™MTAreactionmixturecanberotatedorshakenforlongertimeifdesired.
3.Preparespincolumnforantibody-Buccutite™MTApurification:
3.1 Inverttheprovidedspincolumn(ComponentD)severaltimestore-sUSPendthesettledgelandremoveanybubbles.
3.2 Snapoffthetipandplacethecolumninawashingtube(2mL,notprovided).Removethecaptoallowtheexcesspackingbuffertodrainbygravitytothetopofthegelbed.Ifcolumndoesnotbegintoflow,pushcapbackintocolumnandremoveitagaintostarttheflow.Discardthedrainedbuffer,andthenplacethecolumnbackintotheWashingTube.However,centrifugeimmediatelyifthecolumnisplacedintoa12x75mmtesttube(notprovided).
3.3 Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
3.4 Apply1-2mL1XPBS(pH7.2-7.4)tothecolumn.AftereachapplicationofPBS,letthebufferdrainoutbygravity,orcentrifugethecolumnfor2minutestoremovethebuffer.Discardthebufferfromthecollectiontube.Repeatthisprocessfor3-4times.
3.5 Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
4.Purifytheantibody-Buccutite™MTAsolution:
4.1 Placethecolumn(fromStep3.5)inacleanCollectingTube(1.5mL,notprovided).Carefullyloadthesample(~105μL,fromStep2.2)directlytothecenterofthecolumn.
4.2 Afterloadingthesample,add5μLof1XPBS(pH7.2-7.4)tomakethetotalvolumeof110μL.Centrifugethecolumnfor5-6minutesat1,000xg,andcollectthesolutionthatcontainsthedesiredantibody-Buccutite™MTAsolution.
5.MakeHRP-antibodyconjugation:
5.1 MakeHRP-Buccutite™FOLsolutionbyadding50μLddH2OintothevialofHRP-Buccutite™FOL(ComponentA),mixwellbyrepeatedlypipettingforafewtimesorvortexthevialforafewseconds.
5.2 MixwholevialofHRP-Buccutite™FOLsolution(fromStep5.1)intothepurifiedantibody-Buccutite™MTAsolution(fromStep4.2),mixwellandrotatingthemixturefor1houratroomtemperature.
5.3 TheHRP-antibodyconjugateisnowreadytouse.
Note1:Forimmediateuse,theHRP-antibodyconjugateneedbedilutedwiththebufferofyourchoice.
Note2:Forlongertermstorage,HRP-antibodyconjugatesolutionneedbeconcentratedorfreezedried.
| References&Citations |  PrinterFriendlyVersion |
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4. TijssenP,KurstakE.(1984)Highlyefficientandsimplemethodsforthepreparationofperoxidaseandactiveperoxidase-antibodyconjugatesforenzymeimmunoassays.AnalBiochem,136,451.
5. BoorsmaDM,CuelloAC,vanLeeuwenFW.(1982)Directimmunocytochemistrywithahorseradishperoxidase-conjugatedmonoclonalantibodyagainstsubstanceP.JHistochemCytochem,30,1211.
6. TougardC,Tixier-VidalA,AvrameasS.(1979)Comparisonbetweenperoxidase-conjugatedantigenorantibodyandperoxidase-anti-peroxidasecomplexinapostembeddingprocedure.JHistochemCytochem,27,1630.
7. SternbergerLA,PetraliJP.(1977)Theunlabeledantibodyenzymemethod.Attempteduseofperoxidase-conjugatedantigenasthethirdlayerinthetechnique.JHistochemCytochem,25,1036.
8. BroorsmaDM,SteefkerkJG,KorsN.(1976)PeroxidaseandfluoresceinisothiocyanateasantibodyMarkers.Aquantitativecomparisonoftwoperoxidaseconjugatespreparedwithglutaraldehydeorperiodateandafluoresceinconjugate.JHistochemCytochem,24,1017.
